Nutrient medium for producing cultures of bacteroids of micro-organisms.



IIETTTEE STATES PATENT OFFICE.

RICHARD HARTLEB, OF AAGI-IEN, GERMANY, ASSIGN OR TO OHEMISCIIE FABRIKRHENANIA, OF SAME PLACE.

NUTRIENT MEDIUM FOR PRODUCING CULTURES OF BACTEROIDS OFMICRO-ORGAIIISMS.

SPECIFICATION forming part of Letters Patent N 0. 674,764, dated May 21,1901.

Application filed January 18, 1901- To (tZZ whom it may concern:

Be it known that I, RICHARD HARTLEB, a citizen of Germany, residing atBotanisches Institute der Hochsschule, Aachen,in the Empire of Germany,have invented certain new and useful Nutrient Media for ProducingGultures of Bacteroids of Micro-Organisms, (for which I have applied fora patent in Germany, dated July 18, 1899,) of which the following is aspecification.

This invention relates to the cultivation of bacteroids of the microbesof leguminosze or their permanent forms.

It is known that by inoculating arable soil with artificially-cultivatedleguminosze microbes the growth of plants of the leguminons family ofplants is favorably influenced, the harvest is increased, and the soilis enriched in nitrogen. In this manner sterile soils may be raised to ahigh degree of fertility by growing leguminosze crops without thenecessity for nitrogenous man uring. It is not the bacteria hithertoused for inoculation that induce the infection and the growth of theplant by the formation of nodules and assimilation of nitrogen andenrich the soil in nitrogen, but the bacteroids (which constitute astage of development between the bacteria and their spores) and thepermanent forms of these bacteroids which are the true spores. If,therefore, the conditions for developing the bacteroids and theirpermanent forms do not supervene, the object of inoculating with thebacteria is not achieved and the development of the plants may even beinjured. I have succeeded in producing at will a culture of thesebacteroids and their permanent forms outside the plant by transferringthe organisms of the leguminosee to and further cultivating them in anutrient medium which is characterized by the presence of an acidphosphate of one of the alkalies or alkali earths, a proteid or otherorganic compound of nitrogen, and a carbohydrate or othernon-nitrogenous compound of carbon. The transference of themicro-organism to the above medium may be-made at any stage ofdevelopment. The culture succeeds rapidly both in liquid and in solidnutrients, provided they contain but a small proportion Serial No.43,761. (No specimens.)

of an acid phosphate of one of the alkalies or alkali earths and aproteid I such as potassium,sodium,or calcium acid, legumin phosphate,and a source of carbon, such as grapesugar, a form of starch, inulin,dextrin, arabinose, mannite, or some other carbohydrate. Instead oflegumin there may be used as a source of nitrogen conglutin,gluten-casein, asparagine, leucine, or even ammonium salts or peptone.Less suitable for the purpose are nitrates. The legumin,conglutin, &c.,are obtained in known manner from the shelled and ground seed or germsand are dissolved in an alkali or alkali-earth acid phosphate solution,or they may be dissolved in a neutral solution of phosphate of an alkalior alkali earth. In the latter case phosphoric acid is added, and thislegumin-phosphate solution is added to the other ingredients of thenutrient medium. A productive development of the on ganism is alsoobtainable on solid phosphatic agar-agar culture media by addition ofthe above-named proteid as a source of carbon and nitrogen. In such asolid or liquid medium there is produced a normal develop mentofbacteroids and theirpermanent forms in three to five days even if theculture be months old.

The following receipt is one suitable for a solid nutrient medium forthis invention: dissolve in one liter of water ten to fifteen grams ofagar-agar, two-tenths to one gram of legumin-acid potassium phosphate,obtained as above described, and one to five grams of grape-sugar.

Gelatin cannot be substituted for agaragar, as it is quite useless forthe normal culture of the microbes of leguminosae.

For the liquid nutrient medium the following receipt will generallysufice: dissolve in one liter of water one-tenth to one gram of legumin-acid potassium phosphate, one to five grains of grape-sugar, and tracesof calcium chlorid and magnesium sulfate.

The percentage composition of these nutrient media here given is only byway of example. They must be varied as the kind and the vital force ofthe organism in question may require.

The essential condition for the growth of I00 the culture is the use ofan acid phosphate of one of the alkalies or alkali earths and a proteidor other equivalent compound.

Having thus described the nature of this invention and the best means Iknow of carrying the same into practical effect, I elaim-.

1. A nutrient medium for producing cultures of bacteroids ofmicro-organisms, containing an acid phosphate of one of the alkalies oralkali earths, a proteid, and a carbohydrate.

2.'A nutrientamedium for producing cultures of baeteroids ofmicro-organisms, containing legumin-aeid potassium phosphate andgrape-sugar.

In testimony whereof I have hereunto set my hand in presence of twosuseribing Wi13-' IIGSSBS.

RICHARD HARTLEB. Witnesses:

FRITZ STOLLEWERK, G. SooTT.

